This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. OVERVIEW ***Please note the Tables and Figures mentioned below would not reproduce in this format. Please see attachments sent with the paper copy.*** Core B served the VCII both by providing services to individual VCII laboratories (see Utilization and also Publications) as well as by playing a central role in bringing emerging technologies that are important for VCII investigators (Emerging Technologies). General background on the Microarray Facility is also included (General Background Information). UTILIZATION Services provided to individual VCII labs amounted to 65 Affymetrix GeneChips and 25 hours of bioinformatics service (Table I, Figure 1). Table I Services provided to individual laboratories. 'Hours'refers to bioinformatics service. Figure 1 GeneChip (left) and RNA assessment services (right) provided to VCII laboratories as a percentage of total services. Total utilization of the Microarray Facility consisted of (see also Figure 2) -Experimental design consultations with 18 investigators -Completion of 19 microarray projects (277 GeneChips) -Performed 163 RNA assessments: 79 PicoChips, 81 NanoChips, 3 small RNA's chip (new service, implemented, August, 2008) PUBLICATIONS Publications by VCII Faculty on VCII-related projects that were supported by Core B: 1. Dienz, O., Eaton, S.M., Bond, J.P., Neveu, W., Moquin, D., Noubade, R., Briso, E.M. Charland, C., Leonard, W.J., Ciliberto, G., Teuscher, C., Haynes, L., and Rincon, M. (2009) " The induction of antibody production by IL-6 is indirectly mediated by IL-21 produced by CD4 T cells". J Exp Med. 2009 Jan 16;206(1):69-78. Epub 2009 Jan 12. 2: Lu C, Pelech S, Zhang H, Bond J, Spach K, Noubade R, Blankenhorn EP, Teuscher C. Pertussis toxin induces angiogenesis in brain microvascular endothelial cells. J Neurosci Res. 2008 Sep;86(12):2624-40. PMID: 18500752 [PubMed - in process] EMERGING TECHNOLOGIES Emerging technologies, particularly Affymetrix Exon and Gene arrays as well as next generation sequencing are important for VCII research programs, for example, the VCII external advisory committee (EAC) made a firm recommendation that we provide next generation sequencing technlogy to the VCII. Next generation sequencing Core B is heavily involved in efforts to bring next generation sequencing to UVM (Table 2). Tim Hunter of Core B initiated the process by inviting speakers to UVM. Dr. Budd suggested we apply for an NCRR instrumentation award. The INBRE-funded Vermont Genetics Network took on a leadership role in this process. Table 2. Events related to bringing next generation sequencing technology to UVM. Affymetrix exon and gene arrays Affymetrix exon and gene arrays are emerging as a replacement for Affymetrix 3'arrays. Tim Hunter and Scott Tighe brought these arrays to UVM. Scott Tighe identified a software platform (BioTique Systems XRAY) that supports this technology. The Vermont Genetics Network provided a part time bioinformatics analyst (Dr. Julie Dragon) who is in charge of the exon/gene array pipeline. Dr. Bond is developing an R/Bioconductor pipeline that will give our exon and gene array pipeline the flexibility that we provide for 3'arrays. Network Analysis Core B played a leadership role in bringing Ingenuity IPA to UVM. We evaluated products, coordinated the purchase, and offered special workshops for VCII laboratories. GENERAL BACKGROUND INFORMATION Staff Name Role VCII FTE Bond, Jeffrey Bioinformatics Facility Manager 0.1 Hunter, Timothy Microarray Facility Manager 0.1 Tighe, Scott Research Technician 0.25 Services Services Offered: -Integrated Approaches *Experimental design consultation Assistance in RNA and DNA Extractions *LCM, Flow sorted, difficult tissues -RNA Quality/Quantity assessment -Gene Expression Profiling -DNA Mapping (SNP, LOH, CNV) -Expression profiling using 3', Exon, and Gene Arrays -Hypothesis testing: gene or pathway-based -Support letters for grant applications;text for publication -Discounted pricing on many consumables and reagents through Core Facility negotiations Scientific or technical changes: -Implementation of NuGEN Ovation and Pico WT system for target prep. +Requires minimal RNA input +Useful for FACS sorted, Micro-dissected, and exon application input +Faster turn around time -Ability to assess integrity of small RNA's and miRNA's -Addition of Qubit Spectrofluorometer for accurate quantification -Implementation of Exon and Gene Arrays using NuGEN and AmpTec reagent -New software for Exon analysis: Biotique Systems XRAY -Scott Tighe established protocol for extracting intact RNA from flow sorted cells. Collaborative protocol from roundtable S.Tighe chaired at NERLSCD meeting 2006, Decontaminations procedures, RNase reduction steps, extraction techniques, and QA/QC of RNA Current developmental projects underway by the Microarray Facility: [unreadable]Collaborative effort to develop stand alone software package for QA/QC of 3', Exon, and Gene arrays with Scott Tighe from UVM and John Burke at X-Ray Boutique [unreadable]Comprehensive evaluation of current target preparations on market for working with slightly to severely degraded RNA for 3', gene, and exon arrays. [unreadable] Methods to re-synthesize sense RNA for microarray from cDNA constructed without a T7 promoter -Evaluation of Modified to Amp-Tec reagents -Evaluation of sRNA reagents from Epicenter -Evaluation of SenseAMP from Genisphere 4D Professional Development: Staff attended and participated at the following meetings: -Scott Tighe, Annual Affymetrix Users Meeting, Memphis, TN, Feb. 2009 -Tim Hunter and Scott Tighe: Advances in Microarray Technology, Visited Dr. Herbert Auer's lab, collaboration on DNA Mapping, Barcelona, ESP, May, 2008 -Tim Hunter and Scott Tighe: Association of Biomolecular Resource Facilities, Memphis, TN, 2009 Facility Promotion: -Hosted Open House, June 15, 2008. Over 100 attendees -Provided Workshop on sample requirements and handling for a successful array experiment, June 15, 2008 -Hosted Seminar: "The Challenges and Successes of Implementing Next Generation Sequencing in a Share Resource Facility" June, 2008, Agnes Viale, Director of Genomics Core Laboratory, Memorial Sloan Kettering Cancer Center -Hosted Technology Seminar: "Micro RNA profiling using miScript. Presented by Doug Last, Qiagen. March 14, 2008 -Hosted Technology Seminar, Selective mRNA Amplification using AmpTec Priming Strategies, Presented by Dr. Guido Krupp, August 23, 2008 Facility Presentations: Invited Speaker: -Tim Hunter, (2008) National IDeA States of Biomedical Research Excellence, co-moderator of Core Technology Breakout Session, Washington DC, August, 2008 -Tim Hunter, Northeast Regional Life Sciences Core Directors, "Leveraging Regional Resources", Burlington, VT, Oct., 2008 -Tim Hunter, NCRR Clinical and Translational Informatics Networking Meeting, "Vermont Genetics Network Searchable Core Database", Scottsdale, AZ, Feb., 2009 -Scott Tighe, (2009) Association of Biomolecular Resource Facilities, "Common Practices for Routine RNA Handling: A Three Part Story", Memphis, TN, Feb., 2009 Invited Interview: -Tim Hunter, (2008): "Real-Time PCR: Staying Power", Biotechniques Vol 44, No.2: pp179-183 (Feb 2008) Meeting presentations (Poster): -Scott Tighe and Tim Hunter, Northeast Regional Life Sciences Core Directors, Oct. 2008, Burlington, VT, Vermont Genetics Network Microarray Facility ***Please see attachment for tables and figures: would not reproduce here.***